Cyclic peptides are a large class of secondary metabolites, many of which are important antibiotics (penicillin), immunosuppressants (cyclosporin), or toxins (Amanita toxins). The cyclic tetrapeptide HC-toxin is made by the filamentous fungus Cochliobolus carbonum, a pathogen of maize. HC-toxin is essential for pathogenicity of this fungus. Its structure is cyclo(D-Pro-D-Ala-L-Ala-L-Aeo), where Aeo is 2-amino-9,10-epoxi-8-oxodeanoic acid. We have been studying the biochemistry and molecular genetics of biosynthesis of HC-toxin. We also study the mode of action of HC-toxin and the basis of its specificity against maize. In the course of these experiments we sometimes need chemical analysis of HC-toxin and its natural and synthetic derivatives. For example, maize detoxifies HC-toxin by reducing a critical carbonyl group, and in 1992 the mass spec facility helped us determine the structure of this metabolite. In 1996, we were analyzing the production of HC-toxin by a series of mutants of C. carbonum. These mutants have chromosomal arrangements of their genes devoted to HC-toxin production. Some produce variable amounts of toxin. In particular, one mutant has a prominent second peak of a compound containing D-alanine (determined by feeding 14C-D-alanine to the fungus) in addition to the major form of HC-toxin. It was important to know if this compound could be a shunt metabolite or precursor of HC-toxin, which could help us elucidate the biosynthetic pathway of HC-toxin. The mass spec facility analyzed this compound for us and showed that it had a mass 16 units larger than native HC-toxin. Based also on other criteria, we decided that the compound was probably hydroxy-proline HC-toxin, which had earlier been described as a minor toxin species.